The intracelluar passage of B12 (or cobalamin), a rare and reactive organometallic cofactor, is fraught with perils as it navigates its way to only two enzymes in humans that rely on it: cytoplasmic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). A squad of dedicated chaperones guards against inadvertant loss of B12 via its dilution into solution. The existence of B12 chaperones was first hinted at by clinical genetics studies on patients with inborn errors of B12 metabolism, which lead to isolated or combined homocystinuria and methylmalonic aciduria, and can present early (birth to a few months) or late (years), depending on the severity of the biochemical deficit. The P.I's laboratory has been at the forefront of deciphering functions as the genes encoding B12 chaperones have been identified, and has furnished a wealth of kinetic, spectroscopic and structural insights that are consistently interwoven with clinical data and characterization of patient mutations. In the next cycle, we propose to elucidate how redox-linked coordination chemistry is used as an exquisite and recurring strategy for controlling the chemical reactivity of B12 as it is processed, and for enabling its mobility, as it is transferred between active sites. Specifically, we will address the following aims. (i) Elucidate the cytoplasmic pathway comprising CblC, that displays a ?jack of all trades? style chemical versatility as it processes varied incoming B12 derivatives to a common cob(II)alamin intermediate, and CblD, an elusive protein bearing strong structural resemblance to CblC, but lacking its ability to bind B12. The cytoplasmic pathway terminates in MS and we will address how B12 is loaded from a novel CblD-thiolato-Co(II)-CblC intermediate that we have discovered, and assess the dependence of this transfer process on yet another chaperone, MS reductase. (ii) Elucidate the mitochondrial pathway comprising the enzyme, adenosyltransferase, which doubles as an escort, synthesizing the active 5-deoxyadenosylcobalamin form of the cofactor and transferring it to MCM in a process that is gated by CblA, a GTPase. We will build on our discovery of an unprecedented strategy for cofactor retention involving sacrificial cobalt-carbon bond homolysis that is triggered when MCM acceptor sites are unavailable, and elucidate the rationale for equilibrating MCM-G-protein oligomeric complexes that are nucleotide sensitive. (iii) Elucidate the intersections between B12 and itaconate, an immunomodulatory molecule that is linked to the recently de-orphaned citramalyl-CoA lyase. Itaconyl-CoA (the dehydrated form of citramalyl-CoA) is a potent inhibitor of human MCM. We will determine the underlying mechanism of B12 deficiency when citramalyl-CoA lyase is missing (as in ~3-6% of some populations) and whether mycobacterial MCM represents an additional target of itaconate for shutting down pathogenic cholesterol-dependent energy metabolism.